U.S. patent application number 15/318122 was filed with the patent office on 2017-04-20 for radiolabelling method.
The applicant listed for this patent is GE Healthcare Limited. Invention is credited to Steven Michael Fairway, Matthias Eberhard Glaser, Julian Grigg, IMTIAZ Ahmed KHAN, Ian NEWINGTON, Gareth Edwin Smith, Duncan George Wynn.
Application Number | 20170106104 15/318122 |
Document ID | / |
Family ID | 51410330 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170106104 |
Kind Code |
A1 |
Wynn; Duncan George ; et
al. |
April 20, 2017 |
RADIOLABELLING METHOD
Abstract
The present invention relates to the field of
radiopharmaceuticals for in vivo imaging, in particular to
automated methods for the preparation and purification of
.sup.18F-labelled tau imaging radiotracers. Also provided are
interchangeable cassettes useful in the methods, and the use of
automated synthesizers and cassettes in the methods.
Inventors: |
Wynn; Duncan George;
(Amersham, GB) ; Fairway; Steven Michael; (Oslo,
NO) ; Glaser; Matthias Eberhard; (Amersham, GB)
; NEWINGTON; Ian; (High Wycombe, GB) ; Smith;
Gareth Edwin; (Amersham, GB) ; KHAN; IMTIAZ
Ahmed; (Amersham, GB) ; Grigg; Julian;
(Amersham, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GE Healthcare Limited |
Buckinghamshire |
|
GB |
|
|
Family ID: |
51410330 |
Appl. No.: |
15/318122 |
Filed: |
December 16, 2014 |
PCT Filed: |
December 16, 2014 |
PCT NO: |
PCT/EP2014/078043 |
371 Date: |
December 12, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 15/325 20130101;
B01J 2219/24 20130101; C07D 215/20 20130101; A61P 43/00 20180101;
A61K 51/0455 20130101; B01J 19/24 20130101; C07D 401/04
20130101 |
International
Class: |
A61K 51/04 20060101
A61K051/04; B01D 15/32 20060101 B01D015/32; B01J 19/24 20060101
B01J019/24; C07D 215/20 20060101 C07D215/20; C07D 401/04 20060101
C07D401/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2014 |
GB |
1411571.1 |
Claims
1. An automated method of preparation of an .sup.18F-labelled
radiotracer of Formula (II), which comprises: (i) provision of an
automated synthesizer apparatus which comprises a microprocessor,
and an interchangeable, disposable cassette which comprises a
reaction vessel, a supply of solvents suitable for the preparation
and purification of said radiotracer, and a supply of the precursor
of Formula (I): ##STR00034## (ii) microprocessor-controlled
transfer of said precursor of Formula (I) from step (i) to said
reaction vessel, followed by reaction of said precursor with
[.sup.18F]-fluoride in a suitable solvent, and removal of the
Pg.sup.1 protecting group, to give the .sup.18F-labelled
radiotracer of Formula (II): ##STR00035## wherein: A is chosen
from: ##STR00036## X.sup.1 and X.sup.2 are independently an X.sup.a
or an X.sup.b group; X.sup.3 is an X.sup.a or an X.sup.c group;
X.sup.a is --NR.sup.1R.sup.2; X.sup.b is ##STR00037## X.sup.c is
##STR00038## R.sup.1 and R.sup.2 independently comprise H or
C.sub.1-4 alkyl, or R.sup.1 and R.sup.2 together with the N atom
and optionally the phenyl ring to which they are attached comprise
a 5- or 6-membered nitrogen-containing aliphatic or heteroaromatic
ring, optionally incorporating one further heteroatom chosen from
--O--, --S--, .dbd.N-- and --NR.sup.a--, where R.sup.a is H or
C.sub.1-4 alkyl; R.sup.3 is C.sub.1-4 alkyl, C.sub.1-4 haloalkyl,
C.sub.5-8 aryl or C.sub.6-12 aralkyl; Pg.sup.1 is an alcohol
protecting group; provided that in Formula (I), one X.sup.b group
is present, and in Formula (II) one X.sup.c group is present.
2. The method of claim 1, where step (ii) is carried out by: (a)
reaction of the precursor of Formula (I) with [.sup.18F]-fluoride
in a suitable solvent, to give an .sup.18F-labelled intermediate of
Formula (III): ##STR00039## wherein A.sup.1 is chosen from:
##STR00040## X.sup.4 and X.sup.5 are each independently an X.sup.a
or X.sup.d group; where X.sup.d is: ##STR00041## provided that, in
Formula (III) one X.sup.d group is present; then:-- (b) removal of
the Pg.sup.1 protecting group from said intermediate to give the
.sup.18F-labelled radiotracer of Formula (II).
3. The method of claim 2, where X.sup.2 is X.sup.b, such that the
precursor is of Formula (IA): ##STR00042## and the radiotracer
product is of Formula IIA: ##STR00043## where A.sup.2 is chosen
from: ##STR00044## where X.sup.a is as defined.
4. The method of claim 3, where the precursor is the S-enantiomer
of Formula (IB): ##STR00045## and the radiotracer product is the
S-enantiomer of Formula (IIB): ##STR00046##
5. The method of claim 4, where A is an A.sup.2 group of formula:
##STR00047##
6. The method of claim 5, where --NR.sup.1R.sup.2 is --NHCH.sub.3
or --N(CH.sub.3).sub.2.
7. The method of claim 6, where the cassette further comprises one
to three C18-reverse phase solid phase extraction (SPE) columns,
and said method further comprises step (iii): (iii)
microprocessor-controlled SPE purification of the .sup.18F-labelled
radiotracer of Formula (II) from step (ii) using said cassette SPE
columns, and the solvent(s) of said cassette.
8. The method of claim 7, where the C18-reverse phase SPE column is
C18-silica.
9. The method of claim 8, which is carried out at 15 to 40.degree.
C. with three SPE columns.
10. The method of claim 9, where the SPE columns are first eluted
with an aqueous, water-miscible organic solvent to remove
impurities, and then eluted with ethanol to elute the radiotracer
of Formula (II).
11. The method of claim 10, which further comprises: (iv)
optionally diluting the purified [.sup.18F]-radiotracer of Formula
(II) from step (iii) with a biocompatible carrier; (v) aseptic
filtration of the optionally diluted solution from step (iv) to
give a radiopharmaceutical composition comprising said
radiotracer.
12. A method of purification of the .sup.18F-labelled radiotracer
of Formula (II), (IIA) or (IIB) as defined in claim 1, which
comprises the SPE purification method.
13. A cassette as defined in claim 1.
14. Use of the automated synthesizer apparatus as defined in claim
1, to carry out the method of preparation, or the method of
purification.
15. Use of the cassette of claim 13, to carry out the method of
preparation, or the method of purification.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of
radiopharmaceuticals for in vivo imaging, in particular to
automated methods for the preparation and purification of
.sup.18F-labelled tau imaging radiotracers. Also provided are
interchangeable cassettes useful in the methods, and the use of
automated synthesizers and cassettes in the methods.
BACKGROUND TO THE INVENTION
[0002] Tau is a phosphoprotein having a physiological function of
binding to tubulin to stabilise microtubules. The degree of tau
phosphorylation determines the binding affinity to microtubules-tau
hyperphosphorylation leads to weaker microtubule binding. There is
growing evidence that tau malfunction is implicated in, or triggers
neurodegeneration and dementia. There is therefore significant
interest in the molecular imaging of tau in vivo.
[0003] EP 1574500 A1 (BF Research Institute Inc.) discloses
diagnostic probes for Tau proteins which comprise optionally
radiolabelled compounds of structure:
##STR00001##
[0004] wherein: [0005] R.sub.1, R.sub.2, and R.sub.3 independently
are H, Hal, OH, COOH, SO.sub.3H, NH.sub.2, NO.sub.2,
CO--NH--NH.sub.2, C.sub.1-4 alkyl or O--C.sub.1-4 alkyl, wherein
two R.sub.1 groups together, may form a benzene ring; [0006]
R.sub.4 and R.sub.5 are independently H or C.sub.1-4 alkyl; and
[0007] m and n are independently integers of value 0 to 4.
[0008] WO 2012/067863 discloses that quinolines can be
radiolabelled with radioisotopes suitable for PET or SPECT imaging
to provide Tau imaging agents. WO 2012/067863 mentions that
automated methods optionally including cassettes can be used, but
does not describe particular precursors, methods or cassettes.
[0009] WO 2012/057312 A1 discloses Tau imaging radiotracers which
are radiolabelled compounds of Formula (I):
##STR00002##
wherein
[0010] A is
##STR00003##
R.sup.1 is Hal, a --C(.dbd.O)-lower alkyl group (said alkyl group
may be each independently substituted with one or more substituents
selected from the group consisting of NR.sup.aR.sup.b, Hal, and
OH), a lower alkyl group (said alkyl group may be each
independently substituted with one or more substituents selected
from the group consisting of Hal and OH), an --O-lower alkyl group
(said alkyl group may be each independently substituted with one or
more substituents selected from the group consisting of Hal and
OH), or
##STR00004##
wherein
[0011] R.sup.4 and R.sup.5 are each independently H, a lower alkyl
group, or a cycloalkyl group, or R.sup.4, R.sup.5, and the nitrogen
atom to which they are attached are together form a 3- to
8-membered nitrogen-containing aliphatic ring (one or more carbon
atoms constituting said nitrogen-containing aliphatic ring may be
replaced by a N, S or O atom, and when the carbon atom is replaced
by a N atom, said N atom may be substituted with a lower alkyl
group), or
[0012] R.sup.4 and the nitrogen atom to which it is attached,
together with the ring A, form an 8- to 16-membered
nitrogen-containing fused bicyclic ring system (one or more carbon
atoms constituting said nitrogen-containing fused bicyclic ring
system may be replaced by a N, S or O atom, and when the carbon
atom is replaced by a nitrogen atom, said nitrogen atom may be
substituted with a lower alkyl group), and R.sup.5 is H, a lower
alkyl group, or a cycloalkyl group,
[0013] where a solid line intersected with a broken line designates
a linkage with another structural portion in the general formulae
above,
[0014] R.sup.2 or R.sup.3 is each independently Hal, OH, COOH,
SO.sub.3H, NO.sub.2, SH, NR.sup.aR.sup.b, a lower alkyl group (said
alkyl group may be each independently substituted with one or more
substituents selected from the group consisting of Hal and OH), or
an --O-lower alkyl group (said alkyl group may be each
independently substituted with one or more substituents selected
from the group consisting of Hal and OH),
[0015] the ring A is unsubstituted or substituted with R.sup.6
(wherein R.sup.6 is one or more substituents independently selected
from the group consisting of Hal, OH, COOH, SO.sub.3H, NO.sub.2,
SH, NR.sup.aR.sup.b, a lower alkyl group (said alkyl group may be
each independently substituted with one or more substituents
selected from the group consisting of Hal and OH), and an --O-lower
alkyl group (said alkyl group may be each independently substituted
with one or more substituents selected from the group consisting of
Hal, OH, and an --O-lower alkyl group-O-lower alkyl group (said
alkyl group may be each independently substituted with Hal))),
[0016] R.sup.a and R.sup.b are independently H or a lower alkyl
group (said alkyl group may be each independently substituted with
one or more substituents selected from the group consisting of Hal
and OH),
[0017] m is an integer from 0 to 4, and
[0018] n is an integer from 0 to 4.
[0019] WO 2012/057312 A1 teaches that the .sup.18F-radiotracers are
purified using a combination of Sep-Pak cartridges followed by
semi-preparative HPLC.
[0020] Okamura et al [J. Nucl. Med., 54(8), 1420-1427 (2013)]
disclose that .sup.18F-arylquinolines, in particular
.sup.18F-THK-5105 and .sup.18F-THK-5117 are novel imaging agents
for imaging tau pathology in Alzheimer's disease. Okamura et al use
the following precursors and radiofluorination method:
##STR00005##
Okamura et al use a manual radiolabelling reaction, plus
semi-preparative HPLC for purification of the radiotracer.
[0021] Blom et al [J. Radioanal. Nucl. Chem., 299, 265-270 (2014)]
teach that the radiotracer
##STR00006##
[0022] [.sup.18F]-FMISO, which also incorporates a
fluorohydroxypropyl group, can be prepared via an automated
radiosynthesis. Blom et al studied various solid-phase extraction
(SPE) columns together with the chemical and radiochemical
impurities, and concluded that a hydrophilic-lipophilic balanced
(HLB), polymer-based cartridge was superior to both a mixed mode
(MCX) cartridge and a Sep-Pak C18 cartridge.
[0023] There is therefore still a need for alternative and/or
improved methods of preparing and purifying the tau imaging agents
of WO 2012/057312 A1 and Okamura et al.
The Present Invention
[0024] The precursor synthesis method of the present invention
provides an automated synthesis of quinoline-based
[.sup.18F]-labelled tau radiotracers. The automated method includes
an automated purification methodology, which uses only solid-phase
extraction (SPE) avoids the need for HPLC as taught by the prior
art. The purification method is thus fast (ensuring minimal loss of
tracer due to radioactive decay), and reproducible. The
purification method has also been adapted to work effectively
across the wide range of operating temperatures (ca. 15-37.degree.
C.) that may be found in practice in hot cells where
radiosynthesizer apparatus is located.
[0025] The method comprises the use of an interchangeable,
single-use cassette which is adapted to make the radiosynthesis
even more convenient for the operator, since minimal operator
intervention is required. The cassette approach also has the
advantages of: simplified set-up hence reduced risk of operator
error; improved GMP (Good Manufacturing Practice) compliance;
multi-tracer capability; rapid change between production runs;
pre-run automated diagnostic checking of the cassette and reagents;
automated barcode cross-check of chemical reagents vs the synthesis
to be carried out; reagent traceability; single-use and hence no
risk of cross-contamination, as well as being tamper and abuse
resistance.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In a first aspect, the present invention provides an
automated method of preparation of an .sup.18F-labelled radiotracer
of Formula (II), which comprises: [0027] (i) provision of an
automated synthesizer apparatus which comprises a microprocessor,
and an interchangeable, disposable cassette which comprises a
reaction vessel, a supply of solvents suitable for the preparation
and purification of said radiotracer, and a supply of the precursor
of Formula (I):
[0027] ##STR00007## [0028] (ii) microprocessor-controlled transfer
of said precursor of Formula (I) from step (i) to said reaction
vessel, followed by reaction of said precursor with
[.sup.18F]-fluoride in a suitable solvent, and removal of the
Pg.sup.1 protecting group, to give the .sup.18F-labelled
radiotracer of Formula (II):
##STR00008##
[0028] wherein: [0029] A is chosen from:
[0029] ##STR00009## [0030] X.sup.1 and X.sup.2 are independently an
X.sup.a or an X.sup.b group; [0031] X.sup.3 is an X.sup.a or an
X.sup.c group; [0032] X.sup.a is --NR.sup.1R.sup.2; [0033] X.sup.b
is
[0033] ##STR00010## [0034] X.sup.c is
[0034] ##STR00011## [0035] R.sup.1 and R.sup.2 independently
comprise H or C.sub.1-4 alkyl, or R.sup.1 and R.sup.2 together with
the N atom and optionally the phenyl ring to which they are
attached comprise a 5- or 6-membered nitrogen-containing aliphatic
or heteroaromatic ring, optionally incorporating one further
heteroatom chosen from --O--, --S--, .dbd.N-- and --NR.sup.a--,
where R.sup.a is H or C.sub.1-4 alkyl; [0036] R.sup.3 is C.sub.1-4
alkyl, C.sub.1-4 haloalkyl, C.sub.5-8 aryl or C.sub.6-12 aralkyl;
[0037] Pg.sup.1 is an alcohol protecting group; provided that in
Formula (I), one X.sup.b group is present, and in Formula (II), one
X.sup.c group is present.
[0038] Thus, in the method of the first aspect, the X.sup.b group
of the precursor of Formula (I) contains a reactive site (sulfonate
ester group), which undergoes nucleophilic radiofluorination with
[.sup.18F]-fluoride ion in step (ii) to give the corresponding
X.sup.c substituent of the radiotracer product of Formula (II). The
microprocessor control of step (ii) is achieved via the
microprocessor of said automated synthesizer apparatus. The
provisos of one X.sup.b or X.sup.c group being present imply
that:
[0039] in Formula (I), one of X.sup.1 and X.sup.2 is an X.sup.a
group and the other is an X.sup.b group;
[0040] in Formula (II), one of X.sup.1 and X.sup.3 is an X.sup.a
group and the other is an X.sup.c group;
[0041] The term "radiotracer" has its' conventional meaning and
refers to a radiopharmaceutical used to trace a physiological or
biological process without affecting it. The term
"radiopharmaceutical" has its' conventional meaning and refers to a
radiolabelled compound administered to the mammalian body in vivo
for the purpose of imaging or therapy.
[0042] By the term "automated synthesizer" is meant an automated
module based on the principle of unit operations as described by
Satyamurthy et al [Clin. Positr. Imag., 2(5), 233-253 (1999)]. The
term `unit operations` means that complex processes are reduced to
a series of simple operations or reactions, which can be applied to
a range of materials. Such automated synthesizers are preferred for
the method of the present invention especially when a
radiopharmaceutical composition is desired. They are commercially
available from a range of suppliers [Satyamurthy et al, above],
including: GE Healthcare; CTI Inc; Ion Beam Applications S.A.
(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest
(Germany) and Bioscan (USA).
[0043] Commercial automated synthesizers also provide suitable
containers for the liquid radioactive waste generated as a result
of the radiopharmaceutical preparation. Automated synthesizers are
not typically provided with radiation shielding, since they are
designed to be employed in a suitably configured radioactive work
cell. The radioactive work cell provides suitable radiation
shielding to protect the operator from potential radiation dose, as
well as ventilation to remove chemical and/or radioactive vapours.
The automated synthesizer preferably comprises a cassette. The
automated synthesizer comprises a microprocessor, which controls
the operation of the synthesizer apparatus, including the operation
of any associated cassette.
[0044] By the term "cassette" is meant a unit piece of apparatus
designed such that the whole unit fits removably and
interchangeably onto an automated synthesizer apparatus (as defined
above), in such a way that mechanical movement of moving parts of
the synthesizer controls the operation of the cassette from outside
the cassette, i.e. externally. Suitable cassettes comprise a linear
array of valves, each linked to a port where reagents or vials can
be attached, by either needle puncture of an inverted septum-sealed
vial, or by gas-tight, marrying joints. Each valve has a
male-female joint which interfaces with a corresponding moving arm
of the automated synthesizer. External rotation of the arm thus
controls the opening or closing of the valve when the cassette is
attached to the automated synthesizer. Additional moving parts of
the automated synthesizer are designed to clip onto syringe plunger
tips, and thus raise or depress syringe barrels.
[0045] The cassette is versatile, typically having several
positions where reagents can be attached, and several suitable for
attachment of syringe vials of reagents or chromatography
cartridges (e.g. solid phase extraction or SPE). The cassette
always comprises a reaction vessel. Such reaction vessels are
preferably 1 to 10 cm.sup.3, most preferably 2 to 5 cm.sup.3 in
volume and are configured such that 3 or more ports of the cassette
are connected thereto, to permit transfer of reagents or solvents
from various ports on the cassette. Preferably the cassette has 15
to 40 valves in a linear array, most preferably 20 to 30, with 25
being especially preferred. The valves of the cassette are
preferably each identical, and most preferably are 3-way valves.
The cassettes are designed to be suitable for radiopharmaceutical
manufacture and are therefore manufactured from materials which are
of pharmaceutical grade and ideally also are resistant to
radiolysis.
[0046] Preferred automated synthesizers of the present invention
comprise a disposable or single-use cassette which comprises all
the reagents, reaction vessels and apparatus necessary to carry out
the preparation of a given batch of radiofluorinated
radiopharmaceutical. The cassette means that the automated
synthesizer has the flexibility to be capable of making a variety
of different radiopharmaceuticals with minimal risk of
cross-contamination, by simply changing the cassette. The cassette
approach also has the advantages of simplified set-up hence reduced
risk of operator error; improved GMP (Good Manufacturing Practice)
compliance; multi-tracer capability; rapid change between
production runs; pre-run automated diagnostic checking of the
cassette and reagents; automated barcode cross-check of chemical
reagents vs the synthesis to be carried out; reagent traceability;
single-use and hence no risk of cross-contamination, as well as
being tamper and abuse resistance.
[0047] By the term "precursor" refers to a `radiolabelling
precursor` which means a non-radioactive compound suitable for
reaction with a supply of a radioisotope in a suitable solvent, to
give the radiolabeled compound of interest in the minimum numbers
of steps. Thus, the precursor is designed such that the chemical
and radioactive yield is optimised, and the number of steps
involving the handling of radioactivity is minimized. The precursor
is particularly suitable for radiolabelling with .sup.18F.
[0048] By the term "protecting group" is meant a removable group
which inhibits or suppresses undesirable chemical reactions, and
which is designed such that it can be both attached and removed
to/from the functional group in question under mild enough
conditions that do not modify or compromise the rest of the
molecule. After deprotection the desired product is obtained. The
use of protecting groups is described in Protective Groups in
Organic Synthesis, 4th Edition, Theorodora W. Greene and Peter G.
M. Wuts, [Wiley Blackwell, (2006)]. The term "deprotection" has its
conventional meaning in the field of chemistry and/or
radiochemistry, i.e. the removal of a protecting group.
[0049] The alcohol protecting group (Pg.sup.1) of the first aspect
protects the secondary alcohol group of the X.sup.b group. Suitable
Pg.sup.1 groups include ethers (alkyl, aryl, aralkyl, or silyl);
esters or carbonates. Further details of alcohol protecting groups
are provided by Greene and Wuts (cited above).
[0050] When R.sup.1 and R.sup.2 together with the N atom and
optionally the phenyl ring to which they are attached comprise a 5-
or 6-membered nitrogen-containing aliphatic or heteroaromatic ring,
that means that the 5- or 6-membered ring incorporating one or more
of N, R.sup.1 and R.sup.2 may either be a substituent on the phenyl
ring, or be fused with the phenyl ring bearing --NR.sup.1R.sup.2.
Examples of the former would be piperidine or morpholine rings
singly bonded to the phenyl ring. A preferred example of a fused
ring is when X.sup.a is:
##STR00012##
The X.sup.b group incorporates a sulfonate ester group
--OSO.sub.2R.sup.3. Such sulfonate esters are important leaving
groups in nucleophilic substitution, and the reactivity of the
sulfonate ester towards nucleophilic substitution can be adjusted
depending on the choice of R.sup.3 [M. B. Smith and J. March,
March's Advanced Organic Chemistry, Fifth Edition, John Wiley &
Sons Inc., (2001), pages 445-449].
[0051] The "suitable solvent" for step (ii), includes:
acetonitrile, a C.sub.1-4 alkyl alcohol, dimethylformamide,
tetrahydrofuran, or dimethylsulfoxide, or aqueous mixtures of any
thereof.
Preferred Aspects
[0052] In the method of the first aspect, step (ii) is preferably
carried out by: [0053] (a) reaction of the precursor of Formula (I)
with [.sup.18F]-fluoride in a suitable solvent, to give an
.sup.18F-labelled intermediate of Formula (III):
[0053] ##STR00013## [0054] wherein [0055] A.sup.1 is chosen
from:
[0055] ##STR00014## [0056] X.sup.4 and X.sup.5 are each
independently an X.sup.a or X.sup.d group; [0057] where X.sup.d
is:
[0057] ##STR00015## [0058] provided that, in Formula (III) one
X.sup.d group is present; [0059] then:-- [0060] (b) removal of the
Pg.sup.1 protecting group from said intermediate to give the
.sup.18F-labelled radiotracer of Formula (II).
[0061] In the method of the first aspect, X.sup.2 is preferably
X.sup.b, such that the precursor is of Formula (IA):
##STR00016##
and the radiotracer product is of Formula IIA:
##STR00017##
where A.sup.2 is chosen from:
##STR00018##
In the method of the first aspect, the precursor is more preferably
the S-enantiomeric form of Formula (IB):
##STR00019##
and the radiotracer product is the S-enantiomer of Formula
(IIB):
##STR00020##
The precursor may be enriched in said S-enantiomeric form, to
exceed the 50:50 content of the racemic mixture, and is preferably
in substantially pure form. In the method of the first aspect, A in
Formulae (I), (IA), (IB), (II), (IIA), (IIB) and (III) is
preferably an A.sup.2 group of formula:
##STR00021##
wherein --NR.sup.1R.sup.2 is more preferably --NHCH.sub.3 or
--N(CH.sub.3).sub.2, and most preferably --NHCH.sub.3.
[0062] In the method of the first aspect, Pg.sup.1 is preferably a
Pg.sup.1a group, wherein Pg.sup.1a comprises: [0063] (i) --R.sup.c;
[0064] (ii) --Ar.sup.1; [0065] (iii) --CH(Ar.sup.1).sub.2; [0066]
(iv) --C(Ar.sup.1).sub.3; [0067] (v) tetrahydropyranyl optionally
substituted with one or more substituents chosen from Hal and
OCH.sub.3; [0068] (vi) --CH.sub.2OR.sup.b; [0069] (vii)
--SiR.sup.d.sub.3; [0070] (viii) --(C.dbd.O)R.sup.d; [0071] (ix)
--(C.dbd.O)OR.sup.e wherein R.sup.e is H, R.sup.d, C.sub.1-4
haloalkyl or vinyl; or [0072] (x) --(C.dbd.O)NHR.sup.d; wherein:
[0073] each R.sup.b is independently R.sup.d or C.sub.2-4
alkoxyalkyl optionally substituted with one or more Hal; [0074]
each R.sup.c is independently C.sub.1-4 alkyl; [0075] each R.sup.d
is independently R.sup.c or Ar.sup.1; and [0076] Ar.sup.1 is
independently benzyl or phenyl optionally substituted with one or
more substituents chosen from Hal, CH.sub.3, OCH.sub.3, NO.sub.2 or
--N(CH.sub.3).sub.2. Pg.sup.1 is most preferably
tetrahydropyranyl.
[0077] In the method of the first aspect, R.sup.3 is preferably
chosen from: --CH.sub.3, --CF.sub.3, --C.sub.4F.sub.9,
--CH.sub.2CF.sub.3, --C.sub.6H.sub.4--CH.sub.3,
--C.sub.6H.sub.4--NO.sub.2 or --C.sub.6H.sub.4--Br. R.sup.3 is more
preferably --C.sub.6H.sub.4--CH.sub.3.
[0078] In the method of the first aspect, the cassette preferably
further comprises one to three C18-reverse phase solid phase
extraction (SPE) columns, and said method further comprises step
(iii): [0079] (iii) microprocessor-controlled SPE purification of
the .sup.18F-labelled radiotracer of Formula (II) from step (ii)
using said cassette SPE columns, and the solvent(s) of said
cassette.
[0080] The use of SPE avoids the need for HPLC purification, which
is typically carried out manually, and thus means that the
radiosynthesis and purification of the radiotracer can be carried
out in a fully-automated manner using an appropriate cassette.
Thus, the purification method of the present invention preferably
does not comprise HPLC. In the SPE purification of the first
aspect, the C18-reverse phase SPE column is preferably
silica-based, and is thus preferably a C18-silica SPE column and is
more preferably a tC18+ silica SPE column Polymer-based SPE
cartridges are less preferred, since HLB type cartridges have been
found to bind the radiotracers of the present invention so strongly
that elution becomes difficult. Reverse phase SPE cartridges
suitable for use in the present invention can be obtained from
Waters Limited (730-740 Centennial Court, Centennial Park, Elstree,
Hertfordshire, UK). A suitable size of SPE column for use in the
present invention is 900 mg.
[0081] It is well-established that chromatography such as the SPE
purification process is subject to variations depending on the
ambient temperature. So-called "hot cells" are used for the
production of PET radiotracers. These are enclosures with the
necessary facilities to carry out the radiosynthesis, but also
having radiation-shielding and suitable ventilation to protect the
operator. Such hot cells range from large units that are able to
maintain room temperature (18.degree. C.-22.degree. C.) despite the
large amount of electrical equipment contained within them, to very
small units that can reach operating temperatures of 30.degree.
C.-40.degree. C. The present inventors have found that (see Example
2), elevated temperatures affect the SPE purification such that the
radiotracer product elutes more quickly. As a result, satisfactory
purification of the radiotracer of Formula (II) can be achieved at
temperatures ranges of 15-25.degree. C. using two 900 mg size SPE
columns. At higher temperatures, however, of 15 to 40.degree. C.
three SPE columns are necessary. Hence, the cassette and SPE method
of the first aspect preferably comprises the use of three 900 mg
size SPE columns, since that permits effective purification across
the range of operating temperatures (ca. 15 to 40.degree. C.)
likely to be found in radiosynthesis hot cells. Whilst it is
possible that a smaller number of larger SPE columns could be used,
such larger columns are less likely to be of a size compatible with
automated synthesizer apparatus.
[0082] In the SPE purification of the first aspect, the C18-reverse
phase SPE column is eluted with an elution volume in the range 9-12
mL, preferably 10.5 to 11.5 mL. In the SPE purification method of
the first aspect, the SPE columns are first eluted with an aqueous,
water-miscible organic solvent to remove impurities, and then
eluted with ethanol to elute the radiotracer of Formula (II). The
"aqueous, water-miscible organic solvent" refers to a mixture of
water and the water-miscible organic solvent. Suitable such organic
solvents include acetonitrile, ethanol, THF, isopropanol and
methanol, and are preferably chosen from: acetonitrile, ethanol and
THF, more preferably acetonitrile and ethanol, most preferably
acetonitrile. The aqueous acetonitrile solvent, i.e. the
acetonitrile/water solvent mixture is suitably in the range 20 to
50% v/v, and is preferably in the range 25 to 45%, more preferably
in the range 35 to 40%. 40% aqueous acetonitrile is most
preferred.
[0083] By way of illustration of the SPE purification, the
following discussion refers to Compound 1 and Precursor 1 (see
Scheme 1)--but the same principles apply for other compounds within
the scope of the first aspect.
##STR00022##
[0084] Under the reaction conditions, there is a significant
chemical excess of Precursor 1 over the chemical amount of
[.sup.18F]-fluoride present. Under the reaction conditions,
Precursor 1 also reacts and at least a portion thereof is converted
primarily to the diol (Impurity A; see structures below), and
possibly some of Impurity B. The largest impurity is Impurity A,
which elutes and is removed when the SPE columns are eluted with
aqueous acetonitrile.
[0085] Precursor 1 is significantly more lipophilic than Compound
1, and remains bound to SPE columns--when eluted with either
aqueous acetonitrile or ethanol. Compound 1 does not elute when the
SPE columns are washed with 10-12 mL of aqueous acetonitrile, but
is subsequently eluted when pure ethanol is used to elute the SPE
column(s). In this manner, Compound 1 is purified. Impurity B is
observed less frequently, but any present remains bound to the SPE
column under the conditions of the invention.
[0086] The method of the first aspect preferably further comprises,
in addition to purification step (iii), the following steps:
(iv) optionally diluting the purified [.sup.18F]-radiotracer of
Formula (II) from step (iii) with a biocompatible carrier; (v)
aseptic filtration of the optionally diluted solution from step
(iv) to give a radiopharmaceutical composition comprising said
radiotracer.
[0087] The "biocompatible carrier" is a fluid, especially a liquid,
in which the radioconjugate can be suspended or preferably
dissolved, such that the composition is physiologically tolerable,
i.e. can be administered to the mammalian body without toxicity or
undue discomfort. The biocompatible carrier is suitably an
injectable carrier liquid such as sterile, pyrogen-free water for
injection; an aqueous solution such as saline (which may
advantageously be balanced so that the final product for injection
is isotonic); an aqueous buffer solution comprising a biocompatible
buffering agent (e.g. phosphate buffer); an aqueous solution of one
or more tonicity-adjusting substances (e.g. salts of plasma cations
with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g.
glycerol), or other non-ionic polyol materials (e.g.
polyethyleneglycols, propylene glycols and the like). Preferably
the biocompatible carrier is pyrogen-free water for injection,
isotonic saline or phosphate buffer.
[0088] The "radiopharmaceutical composition" is a pharmaceutical
composition comprising said radiotracer. Such compositions have
their conventional meaning, and in particular are in a form
suitable for mammalian administration, especially via parenteral
injection. By the phrase "in a form suitable for mammalian
administration" is meant a composition which is sterile,
pyrogen-free, lacks compounds which produce toxic or adverse
effects, and is formulated at a biocompatible pH (approximately pH
4.0 to 10.5). Such compositions lack particulates which could risk
causing emboli in vivo, and are formulated so that precipitation
does not occur on contact with biological fluids (e.g. blood). Such
compositions also contain only biologically compatible excipients,
and are preferably isotonic.
[0089] The production of [.sup.18F]-fluoride suitable for
radiopharmaceutical applications is well-known in the art, and has
been reviewed by Hjelstuen et al [Eur. J. Pharm. Biopharm., 78(3),
307-313 (2011)], and Jacobson et al [Curr. Top. Med. Chem., 10(11),
1048-1059 (2010)].
[0090] A non-automated radiosynthesis of Compound 1 has been
reported by Okamura et al [J. Nucl. Med., 54(8), 1420-1427
(2013)].
[0091] Substituted quinolones of Formula (I) can be synthesized by
conventional quinoline syntheses [Kouznetsov et al, Curr. Org.
Chem., 9, 141-161 (2005)]. The syntheses of several
2-arylquinolines has been provided by Tago et al [J. Lab. Comp.
Radiopharm., 57(1), 18-24 (2014)]. Further details of the precursor
syntheses are provided in WO 2012/057312 A1. Thus, WO 2012/057312
A1 discloses the following synthesis of the .sup.18F labelling
precursors having alkoxy substituents at the 6-position
functionalised with hydroxy and .sup.18F groups:
##STR00023## ##STR00024##
[0092] The present supporting Examples provide further experimental
details. The corresponding enantiomers can be obtained by adapting
the synthesis using chiral starting materials, or resolution of the
racemic mixture using e.g. chiral chromatography or crystallisation
of a chiral salt as is known in the art.
[0093] In a second aspect, the present invention provides a method
of purification of the .sup.18F-labelled radiotracer of Formula
(II), (IIA) or (IIB) as defined in the first aspect, which
comprises the SPE purification method as described in a preferred
embodiment of the first aspect.
[0094] Preferred aspects of the radiotracer, precursor and
purification method in the second aspect, are as described in the
first aspect (above).
[0095] In a third aspect, the present invention provides a cassette
as described in the first aspect (above). Preferred aspects of the
cassette in the third aspect are as described in the first aspect
(above).
[0096] In a fourth aspect, the present invention provides the use
of the automated synthesizer apparatus as defined in the first
aspect, to carry out the method of preparation of the first aspect,
or the method of purification of the second aspect. Preferred
aspects of the automated synthesizer apparatus and method in the
fourth aspect, are as described in the first aspect (above).
[0097] In a fifth aspect, the present invention provides the use of
the cassette of the third aspect, to carry out the method of
preparation of the first aspect, or the method of purification of
the second aspect. Preferred aspects of the cassette in the fifth
aspect are as described in the third aspect (above).
BRIEF DESCRIPTION OF THE FIGURES
[0098] FIG. 1 and FIG. 2 illustrate exemplary cassettes of the
invention useful for carrying out particular examples of the method
of the invention.
BRIEF DESCRIPTION OF THE EXAMPLES
[0099] The invention is illustrated by the non-limiting Examples
detailed below. Example 1 provides the synthesis of a
radiolabelling precursor of the invention ("Precursor 2"). Example
2 demonstrates the effect of elevated temperature on the
radiosynthesis and purification of Compound 1. Example 3 provides
an improved synthesis and purification of Compound 1, which is
suitable for use at range of temperatures.
Compounds of the Invention
TABLE-US-00001 [0100] Name Structure Compound 1 ##STR00025##
Precursor 1 ##STR00026## Impurity A ##STR00027## Impurity B
##STR00028## Precursor 2 ##STR00029## Compound 2 ##STR00030##
Precursor 3 ##STR00031## Compound 3 ##STR00032## Precursor 4
##STR00033##
Abbreviations
Ac: Acetyl
Acm: Acetamidomethyl
ACN: Acetonitrile
[0101] AcOH: Acetic acid. Boc: tert-Butyloxycarbonyl tBu:
tertiary-butyl
DCM: Dichloromethane
[0102] DIPEA: N,N-Diisopropylethyl amine
DMF: Dimethylformamide
[0103] EtOAc: ethyl acetate; EtOH: ethanol
DMSO: Dimethylsulfoxide;
GMP: Good Manufacturing Practice;
[0104] HPLC: High performance liquid chromatography; MeCH:
acetonitrile MW: molecular weight; Ms: mesylate i.e. sulfonate
ester of methanesulfonic acid. RCP: radiochemical purity; RCY:
radiochemical yield; RP-HPLC: reverse-phase high performance liquid
chromatography; SPE: solid phase extraction; TBAF:
tetrabutylammonium fluoride; tBu: tert-butyl; TFA: Trifluoroacetic
acid;
THF: Tetrahydrofuran;
[0105] THP: tetrahydropyranyl; TLC: thin layer chromatography;
Trt: Trityl;
[0106] Tf: triflate, i.e. sulfonate ester of
trifluoromethanesulfonic acid. Ts: tosylate, i.e. sulfonate ester
of para-toluenesulfonic acid.
Example 1: Synthesis of Precursor 2
Step (a): 2-(5-Fluoro-2-nitrophenyl)-1,3-dioxolane
[0107] 5-Fluoro-2-nitrobenzaldehyde (14.4 g, 85 mmol),
ethane-1,2-diol (14.48 mL, 260 mmol) and 4-toluenesulfonic acid
monohydrate (0.826 g, 4.34 mmol) were added to toluene (350 mL) and
the mixture heated to reflux under nitrogen with a Dean & Stark
condenser. The reaction was allowed to cool after 4.5 h. After 30
h, the solution was decanted from the dark sticky residue at the
bottom of the flask. Added EtOAc (275 mL) and washed with saturated
aqueous sodium bicarbonate (70 mL), water (140 mL), brine (70 mL)
and passed through a phase separator then evaporated to dryness to
give a dark brown oil (.about.18 g). This was dissolved in
DCM:petrol (3:2) and purified by chromatography on silica gel
eluting with dichloromethane (A): Petroleum ether (B) (60% B, 340
g, 15 CV, 100 mL/min) to give the expected product as a yellow oil
(16.52 g, 91%).
[0108] .sup.1H NMR (400 MHz,) .delta. 8.10-7.95 (dd, J=9.0, 4.9 Hz,
1H, Ar-H3), 7.58-7.44 (dd, J=9.1, 2.9 Hz, 1H, Ar-H4), 7.22-7.10
(ddd, J=9.1, 7.2, 2.9 Hz, 1H, Ar-H6), 6.63-6.41 (s, 1H, OC(O)H) and
4.14-3.96 (dddd, J=14.1, 8.6, 6.8, 3.3 Hz, 4H, 2.times.CH.sub.2).
.sup.13C NMR (101 MHz,) .delta. 164.8 (d, J=259 Hz, C--F), 144.7
(C--NO.sub.2), 137.3 (d, J=8 Hz, Ar-C1), 127.7 (d, J=9 Hz, Ar-C3),
116.5 (d, J=25 Hz, Ar-C4/6), 115.1 (d, J=25 Hz, Ar-C4/6), 99.1
(OCHO) and 65.5 (2.times.CH.sub.2).
Step (b): 3-(1,3-Dioxolan-2-yl)-N-methyl-4-nitroaniline
[0109] 2-(5-Fluoro-2-nitrophenyl)-1,3-dioxolane [Step (a), 5.21 g,
24.44 mmol] was dissolved in ethanol (37 ml) and methylamine (5.5
mL, 33 wt % in ethanol, 46.9 mmol) added. The yellow solution was
stirred at ambient temperature for 10 minutes then heated to reflux
for 18 h, when LCMS and TLC (1:1 DCM:petrol) showed no remaining
starting material. The solution was allowed to cool and evaporated
to dryness, dissolved in DCM (100 mL) and washed with saturated
aqueous sodium bicarbonate (40 mL) then water (2.times.40 mL) and
passed through a phase separator and evaporated to a deep
yellow-orange oil (5.45 g, 99%).
[0110] LCMS calcd for C.sub.10H.sub.12N.sub.2O.sub.4: 224.1; found
225.0 [M+H]+.
[0111] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.05 (d, J=9.0 Hz,
1H, Ar-5H), 6.92 (d, J=2.7 Hz, 1H, Ar-2H), 6.62 (s, 1H, CH), 6.50
(dd, J=9.1, 2.7 Hz, 1H, Ar-6H), 4.59 (br s, 1H, NH), 4.06 (m, 4H,
2.times.CH.sub.2) and 2.94 (d, J=5.1 Hz, 3H, NCH.sub.3). .sup.13C
NMR (101 MHz, CDCl.sub.3) .delta. 153.4 (C--NH), 137.6
(C--NO.sub.2), 136.4 (Ar-3C), 128.7 (Ar-5C), 110.4 (Ar-6C), 109.8
(Ar-2C), 99.9 (CH), 65.3 (2.times.CH.sub.2) and 30.2
(N--CH.sub.3).
Step (c) 5-(Methylamino)-2-nitrobenzaldehyde
[0112] 3-(1,3-Dioxolan-2-yl)-N-methyl-4-nitroaniline [Step (b),
5.45 g, 24.31 mmol] was dissolved in acetone (55 mL) and
hydrochloric acid (1N) (2.00 g, 55 mmol) was added and the yellow
solution heated to 60 C for 3 h, when LCMS and TLC showed no
residual starting material. The solution was cooled and neutralised
with aqueous sodium bicarbonate and extracted into ethyl acetate
(3.times.70 mL). The combined organics were passed through a phase
separator and evaporated to give a yellow solid (4.28 g, 98%).
[0113] LCMS calcd for C.sub.8H.sub.8N.sub.2O.sub.3: 180.1; Found
180.92 [M+H]+.
[0114] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 10.51 (s, 1H,
HC.dbd.O), 8.10 (d, J=9.1 Hz, 1H, Ar-3H), 6.85 (d, J=2.8 Hz, 1H,
Ar-6H), 6.68 (dd, J=9.0, 2.8 Hz, 1H, Ar-4H), 4.83 (br s, 1H, NH)
and 2.98 (d, J=5.1 Hz, 3H, N--CH.sub.3). .sup.13C NMR (101 MHz,
CDCl.sub.3) .delta. 190.2 ({right arrow over (C)}.dbd.O), 153.8
(Ar--CNH), 137.9 (Ar--CNO.sub.2), 135.6 (Ar--CCHO), 128.0 (Ar-3CH),
113.6 (Ar-4CH), 110.9 (Ar-6CH) and 30.3 (N--CH.sub.3).
Step (d) 2-(4-Methoxyphenyl)-N-methylquinolin-6-amine
[0115] 5-(Methylamino)-2-nitrobenzaldehyde [Step (c), 1.39 g, 7.72
mmol] was dissolved in ethanol (40 mL) in a 50 mL borosilicate tube
and iron powder (1.72 g, 30.9 mmol) and hydrochloric acid (3.86 mL,
0.1N, 0.386 mmol) added and the tube sealed with PTFE/silicone
screw-cap and heated in a pre-heated oil-bath at 100 C. After 2 h,
the tube was removed and cooled in water and the pressure carefully
released, when LCMS showed no remaining starting material. Added
1-(4-methoxyphenyl)ethanone (1.16 g, 7.72 mmol) and powdered
potassium hydroxide (0.52 g, 9.26 mmol) to the mixture, resealed
and heated at 100 C for 22 h. Cooled, diluted with water (150 mL)
and extracted with DCM (4.times.50 mL), washed combined organics
with water (50 mL) and passed through a phase separator and
evaporated to give a yellow-brown gum (1.94 g). This was purified
by chromatography on silica gel eluting with petroleum ether (A):
ethyl acetate (B) (10-100% B, 100 g, 15 CV, 85 mL/min) to give a
pale yellow solid (530 mg, 26% yield).
[0116] LCMS calcd for C.sub.17H.sub.16N.sub.2O 264.1; found 265.0
[M+H]+.
[0117] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.12-8.01 (m, 2H,
Ph-H), 7.94 (d, J=8.6 Hz, 1H, Ar--H), 7.90 (d, J=9.1 Hz, 1H, Ar--H)
7.68 (d, J=8.6 Hz, 1H, Ar--H), 7.05 (dd, J=9.0, 2.6 Hz, 1H, Ar--H),
7.01 (m, 2H, Ph-H), 6.66 (d, J=2.5 Hz, 1H, Ar-H), 4.03 (br s, 1H,
NH), 3.58 (s, 3H, OCH.sub.3), 2.90 (s, 3H, NCH.sub.3). .sup.13C NMR
(101 MHz, CDCl.sub.3) .delta. 160.2 (C--OMe), 152.9 (Ar--C--N),
147.0 (C--NMe), 143.2 (Ar-C10), 134.6 (Ar-C-4), 132.9 (Ph-C1),
130.4 (Ar-C7), 128.8 (Ar-C9), 128.4 (Ph-C2&6), 121.4 (Ar-C8),
118.9 (Ar-C3), 114.2 (Ph-C3&5), 102.5 (Ar-C5), 55.5
(O--CH.sub.3) and 30.8 (N--CH.sub.3).
Step (e) 4-(6-(Methylamino)quinolin-2-yl)phenol
[0118] 2-(4-Methoxyphenyl)-N-methylquinolin-6-amine [Step (d), 680
mg, 2.57 mmol] was dissolved in DCM (35 mL) and boron tribromide
(10.3 mL, 1M in DCM, 10.3 mmol) was added and the mixture stirred
for 18 h--some insoluble gum formed--when LCMS showed mainly
desired product with a little residual starting material. Added
methanol (2-3 mL dropwise) to destroy excess BBr.sub.3 and filtered
off the yellow solid. Stirred with saturated aqueous sodium
bicarbonate and filtered. Allowed to dry on filter paper to give
the desired product as a yellow solid (602 mg, 93%).
[0119] LCMS calcd for C.sub.16H.sub.14N.sub.2O 250.1; found 251.0
[M+H]+.
[0120] .sup.1H NMR (400 MHz, d.sub.6-DMSO) .delta. 7.96 (m, 3H),
7.77 (d, J=8.7 Hz, 1H, C8-H), 7.65 (d, J=9.0 Hz, 1H, C4-H), 7.11
(dd, J=9.1, 2.0 Hz, 1H, C3-H), 6.81 (d, J=8.5 Hz, 2H, C2'
&6'-H), 6.59 (m, 1H, C5-H), 6.13 (d, J=4.8 Hz, 1H, NH) and,
2.74 (d, J=4.8 Hz, 3H, N--CH.sub.3). .sup.13C NMR (101 MHz,
d.sub.6-DMSO) .delta. 159.3 (C--OH), 151.6 (C6-N), 148.0 (C9),
142.4 (C4'), 134.6 (C4-H), 129.9 (C7-H), 129.0 (C10), 128.3
(C3-H'& C5'-H), 122.1 (C8-H), 118.4 (C3-H), 116.1 (C2'-H &
C6'-H), 101.2 (C5-H) and 30.3 (N--CH.sub.3).
Step (f)
3-(4-(6-(Methylamino)quinolin-2-yl)phenoxy)-2-((tetrahydro-2H-pyr-
an-2-yl)oxy)propyl 4-methylbenzenesulfonate
[0121] 4-(6-(Methylamino)quinolin-2-yl)phenol [Step (e), 300 mg,
1.2 mmol] and potassium carbonate (215 mg, 1.56 mmol) were mixed in
a 25 mL rb flask fitted with a rubber septum and a nitrogen
balloon. Dry DMF (10 mL) was added followed by
2-((tetrahydro-2H-pyran-2-yl)oxy)propane-1,3-diyl
bis(4-methylbenzenesulfonate) (581 mg, 1.2 mmol) [Oh et al, Nucl.
Med. Biol., 32(8), 899-905 (2005)], and the mixture stirred
vigorously and heated at an external temp of 90 C. Cooled after 22
h, when TLC showed incomplete reaction. Nevertheless, ice water (30
mL) was added and the organic material extracted into ethyl acetate
(3.times.15 mL). Washed the combined organics with water
(2.times.15 mL), brine (15 mL) and passed through a phase separator
and evaporated. TLC (EtOAc:petrol 1:1) and LCMS showed the 2 main
peaks as starting material and product. Adsorbed onto silica from
ethyl acetate and acetonitrile mixture and purified by
chromatography on silica gel eluting with petroleum ether (A):
ethyl acetate (B) (10-100% B, 50 g, 20 CV, 40 mL/min) to give the
major peak being product but contaminated by starting material.
Re-purified by chromatography on silica gel eluting with
dichloromethane (A): ethyl acetate (B) (20-60% B with initial
isocratic at 21%, 25 g, 25 CV, 40 mL/min) to give pure product as a
yellow solid (65 mg, 10%).
[0122] LCMS calcd for C.sub.31H.sub.34N.sub.2O.sub.6S 562.2; found
563.0 [M+H]+.
[0123] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.02 (m, 2H, C3'-H
& C5'-H), 7.96 (d, J=8.6 Hz, 1H, C7-H), 7.90 (d, J=9.1 Hz, 1H),
7.77 (m, 2H), 7.69 (d, J=8.6 Hz, 1H), 7.26 (m, 2H), 7.08 (dd,
J=9.0, 2.6 Hz, 1H), 6.89 (m, 2H), 6.68 (d, J=2.5 Hz, 1H), 4.81 (t,
J=3.3 Hz, 1H), 4.40-3.94 (m, 3H), 2.99-2.89 (s, 1H), 2.37 (s, 3H,
Ar--CH.sub.3), 1.86-1.63 (m, 2H), 1.61-1.44 (m, 3H). .sup.13C NMR
(101 MHz, CDCl.sub.3) .delta. 158.9, 158.8, 152.6, 147.1, 145.0,
143.1, 134.6, 133.3, 132.6, 130.4, 130.0, 129.9, 128.9, 128.3,
128.1, 128.0, 121.5, 118.8, 114.8, 102.4, 99.1, 98.5, 72.7, 72.3,
69.4, 69.1, 66.9, 66.2, 62.9, 62.3, 60.5, 30.8, 30.6, 30.5, 25.4,
21.8, 21.2, 19.5, 19.1 and 14.3.
Example 2: Effect of Temperature on the Automated Radiosynthesis of
Compound 1
[0124] [.sup.18F]-fluoride was produced using a GE PETtrace
cyclotron with a silver target via the [.sup.18O](p,n) [.sup.18F]
nuclear reaction. Total target volumes of 3.2-4.8 mL were used. The
radiofluoride was trapped on a Waters QMA cartridge
(pre-conditioned with potassium carbonate), and the
[.sup.18F]-fluoride was eluted with a solution of TBAF bicarbonate
(0.75 M, 160 .mu.L) in acetonitrile (640 .mu.L). Nitrogen was used
to drive the solution off the QMA cartridge to the reaction vessel.
The [.sup.18F]-fluoride was dried for 9 minutes at 120.degree. C.
under a steady stream of nitrogen and vacuum.
[0125] To investigate the impact of the anticipated PET cell
temperature range on the efficacy of the SPE process,
radiosynthesis studies were conducted at the upper end of the range
(35.degree. C.) with a single Waters tC18+ SPE cartridge.
[0126] A cassette was fitted to a FASTlab synthesiser apparatus (GE
Healthcare). [.sup.18F]Fluoride was transferred via the activity
inlet of the FASTlab cassette using vacuum. The activity was
transferred from the activity inlet to the (pre-treated) QMA
cartridge where the [.sup.18F] was trapped and the water passed
through to the .sup.18O water recovery vial, using a combination of
N2 to push and vacuum to pull. After the transfer of the eluent
containing the .sup.18F-activity into the reaction vessel, the
solvents were evaporated to dryness. The evaporation was carried
out with heating under nitrogen flow and under vacuum.
[0127] Precursor 1 (1.8 mL of a 1.5 mg/mL solution in DMSO) was
added to the dry residue. Nucleophilic substitution at 130.degree.
C. was carried out in the closed reaction vessel, in which the
tosylate group of the precursor was replaced by the .sup.18F-ions.
After labelling, the solution was cooled to 70.degree. C. The
tetrahydropyranylated intermediate was converted into Compound 1 by
removing the THP protecting group. This deprotection was carried
out in the reaction vessel by the addition of aqueous HCl (0.35 mL
of 4M HCL diluted with 0.82 mL of water), heating at 90.degree. C.
for 35 seconds, followed by quenching via the addition of 4%
aqueous ammonia solution (1.4 mL).
[0128] The resulting Compound 1 was obtained in a DMSO/aqueous
mixture, and was adjusted to an 80:20 aqueous: organic mixture,
prior to loading onto two Waters tC18+ SPE cartridges in
series.
[0129] Analysis of fractions of the 40% acetonitrile wash volume
collected from the FASTlab.TM. combined with reduced RCY showed
significant loss of radiotracer. GE FASTlab.TM. log files were used
to determine that very low wash volumes were sufficient to
completely elute all the radiotracer at 35.degree. C.
Example 3: Automated Radiosynthesis of Compound 1
[0130] The radiosynthesis of Example 2 was adapted using a third
Waters tC18+ cartridge added to the GE FASTlab.TM. cassette and
this layout was studied over the temperature range from
19.3.degree. C.- to 37.0.degree. C. FIG. 1 illustrates the cassette
layout used wherein 1 indicates the activity inlet, 2 a buffer
volume, 3 a supply of N.sub.2, each of 4a-j a valve, 5 effluent, 6
is the reaction vessel, 7-10 are reagent positions wherein 7 is
precursor, 8 is 4M HCl, 9 is 4% ammonia, 10 is water and 11 is
vacant. Reference number 12 indicates the three Waters tC18+
cartridges, 13 the product outlet, 14 the waste bottle, 15 40% MeCN
and 16 100% EtOH.
[0131] The resulting Compound 1 was obtained in an
acetonitrile/aqueous mixture, and was adjusted to an 80:20
aqueous:organic mixture, prior to loading onto three Waters tC18+
SPE cartridges in series. The SPE cartridges were then rinsed with
water and washed with 10.6 mL of 40% aqueous acetonitrile to remove
Impurity A prior to elution of Compound 1 with ethanol.
[0132] The Compound 1 obtained had a total chemical content of 5-10
.mu.g/mL and radiochemical purity (RCP) in the range 92 to 97% at a
specific activity of 100-1000 GBq/.mu.mol, for starting .sup.18F
activities in the range 40-60 GBq. In addition, by studying SPE
wash fractions (via collecting the samples and analysing
information provided by the radio detectors in the GE FASTlab.TM.
log file), it was noted that product losses were negligible and
hence good radiochemical yield (RCY) was achieved. Chemical
content, RCY and specific activity measurements were not observed
to be affected by the addition of a third SPE cartridge.
Example 4: Automated Radiosynthesis of Compounds 2 & 3
[0133] The cassette layout of FIG. 2 was used to synthesise
Compounds 2 & 3. In FIG. 2: 1 indicates the activity inlet, 2 a
buffer volume, 3 a supply of N.sub.2, each of 4a-j a valve, 5
effluent, 6 is the reaction vessel, 7-10 are reagent positions
wherein 7 is precursor (Precursor 3 and Precursor 4, respectively
for Compound 2 and Compound 3), 8 is DMSO, 9 is 4M HCl, 10 is water
and 11 is 4% ammonia. Reference number 12 indicates the three
Waters tC18+ cartridges, 13 the product outlet, 14 the waste
bottle, 15 is MeCN (40% and 28.5% for Compound 2 and Compound 3,
respectively) and 16 100% EtOH. Precursors 2 and 4 were obtained
using methods similar to that for Precursor 1 (i.e. as per methods
described in Okamura et al J. Nucl. Med., 54(8), 1420-1427
(2013)).
[0134] For Compound 2, 11 mL of 40% MeCN was required to give a
chemical content of 0.1-1.9 .mu.g/mL over the temperature range
from 21.degree. C.-39.degree. C. A decay corrected yield of 42-57%
was obtained when using 4 mg of precursor. The RCP was >90% when
starting with 60 GBq or less.
[0135] For Compound 3, 11.5 mL of ca. 28.5% MeCN was required to
give a chemical content of <1.0 .mu.g/mL over the temperature
range 20-30.degree. C. Decay corrected yields of 20-25% were
obtained with 3 mg precursor. For this compound, the starting
activity was increased to 100 GBq without any affect of RCP,
showing RCP's of >98%. However, the SPE purification works at a
tighter temperature range as compared to THK5317. At around
25.degree. C. and below, the product is trapped on the 2.sup.nd SPE
cartridge and is eluted into the product vial, whereas at around
26-30.degree. C. the product is trapped on the 3.sup.rd SPE
cartridge before being eluted into the product vial. Above
30.degree. C. some of the product is washed to waste and the
resulting yield is thus decreased. Therefore, the operating
temperature for THK5351 is 20-30.degree. C.
* * * * *